Production of high purity chondroitinase ABC

ABSTRACT

The present invention provides a method for purifying Chondroitinase ABC (ChABC). The present method includes using a heparin-immobilized affinity chromatography column, and through chromatography method obtaining a purified ChABC from a matrix containing said ChABC. The present method is capable of obtaining ChABC in high purity with the advantages of simplicity in preparation and high yield.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. non-provisionalpatent application Ser. No. 15/788,824 filed Oct. 20, 2017, which is adivisional application of U.S. non-provisional patent application Ser.No. 15/494,589 filed Apr. 24, 2017 (now patented under the U.S. Pat. No.9,796,970), and the disclosures of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a process for the purification ofchondroitinase ABC (ChABC), and more particularly to a method forpurifying ChABC from a matrix containing ChABC by affinitychromatography.

BACKGROUND

Chondroitinase ABC (ChABC) is an enzyme that catalyzes chemicalreactions involving the eliminative degradation of polysaccharidescontaining 1,4-beta-D-hexosaminyl and 1,3-beta-D-glucuronosyl or1,3-alpha-L-iduronosyl linkages to disaccharides containing4-deoxy-beta-D-gluc-4-enuronosyl groups. In animal tissues, ChABC actson chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate.Chondroitin sulfate is the most abundant of the glycosaminoglycans inbiological systems and includes a variety of sulfated and non-sulfatedcarbohydrate derivatives including sulfated glucuronic acid,N-acetylgalactosamine, and iduronic acid. These materials are oftenlinked to proteins to form chondroitin sulfate proteoglycans (CSPG),which play a number of structural and functional roles in biologicalsystems (e.g., cell-cell interactions with extracellular matrix,directing or inhibiting neurodevelopment, compositing cartilage tobecome a structural component). The biosynthesis of these CSPG moleculesand the proper location are critical to the normal functioning ofhumans. In fact, many diseases associated with the lack of CSPG,accumulation, or dislocation, such as diffuse Lewy body disease,mucopolysaccharide disease IV-A type and mucopolysaccharide disease VIItype, disc herniation and so on.

Chondroitinase enzymes are a group of enzymes that can be divided intofour types with different activity and substrate specificity(chondroitinase ABC, chondroitinase AC, chondroitinase B andchondroitinase C). These enzymes, particularly chondroitinase ABC, haverecently been shown to be potential new biological agents for thetreatment of many CSPG-related diseases. For example, researchers havefound that ChABC can significantly promote functional recovery of adamaged spinal cord. Other researchers have shown that the injection ofchondroitin sulfate into the affected parts of keloid and/orhypertrophic scars can greatly improve the symptoms. There has also beendeveloped a successful model for the treatment of rabbit disc herniationwith ChABC. Recent scientific studies have suggested that ChABC has morepotential medical applications, including the treatment of amblyopia,nerve and spinal cord injuries, posterior vitreous detachment andinhibition of tumor metastasis.

Whether ChABC can be successfully used as a biotherapeutic agent issignificantly dependent on the production and purification of ChABC.ChABC's existing upstream production techniques are based on thefermentation of unmodified proteus vulgaris or recombinant expressionhosts (e.g., E. coli). By appropriate optimization, both of theseproduction methods can yield a large number of non-purified matricescontaining ChABC enzyme with a host organism. Because the matricescontain substantial quantities of protein, nucleic acid, endotoxin, andother impurities, the development of efficient downstream purificationmethods for obtaining high purity ChABC is critical. Further, the amountand nature of impurities from different expression hosts are verydifferent, so in order to purify ChABC from a particular expressionhost, a specific downstream purification procedure will typically bedeveloped separately, especially when using only non-specificinteraction ion exchange (IEX) or hydrophobic interaction (HIC)chromatography. However, the development and optimization of IEX/HICchromatography is time-consuming and requires considerable manpower andmaterial resources. Consequently, the typical process requires multipleIEX/HIC chromatography steps to achieve high purity target proteinsresulting in high overall cost.

In contrast to IEX/HIC chromatography, affinity chromatography presentsseveral potential advantages. In affinity chromatography, molecules ormolecules that interact specifically with the target protein arepartially immobilized on the chromatography filler. This highlyselective interaction can effectively extract the desired protein andremove impurities. However, affinity chromatography has not been used asa purification tool for ChABC without affinity tag. Because ChABC is anenzyme, it is not easy to design affinity chromatography for nativeChABC. This is because most of the known molecules that can specificallybind to native ChABC are ChABC substrates that will be degraded whencombined with the ChABC enzyme. In other words, if these substratemolecules are immobilized on the stationary phase, they will bedecomposed by the enzyme during the purification process, so that ChABCcannot be captured from the solution.

Thus there is a need in the art for novel purification techniques forChABC to enable cost-effective production of ChABC for use as abiotherapeutic drug.

SUMMARY OF THE INVENTION

In order to solve the problems in the prior art, the inventors havefound that heparin not only specifically binds to ChABC, but is notdegraded at the time of binding. Based on this finding, the presentinvention provides a method for purifying ChABC from a matrix containingChABC by chromatography using an affinity column immobilized withheparin.

In one embodiment, the present invention provides a specific affinitychromatography process for preparing high purity chondroitinase ABC froma matrix including chondroitinase ABC and a microorganism to producechondroitinase ABC. The process comprises providing a matrix of materialcomprising one or more microorganisms to produce chondroitinase ABC, themicroorganism selected from genetically-modified Escherichia coli,genetically-modified Bacillus subtilis, Proteus vulgaris, Pichiapastoris, and/or Saccharomyces cerevisiae and chondroitinase ABCproduced by the microorganism. Cell debris are removed by centrifugationand adjusting the pH and conductivity of the matrix material.

A specific affinity heparin-immobilized chromatography column isprovided comprising heparin immobilized on a resin, wherein theheparin-immobilized chromatography column is pre-equilibrated to a pH ofapproximately pH 7.0-7.5. The heparin-immobilized chromatography columnis loaded with the pH- and conductivity-adjusted matrix material. Theheparin-immobilized chromatography column loaded with matrix material iswashed with washing liquid in two or more washes at a first pH and asecond pH wherein the first pH is higher than the second pH to washunwanted protein, DNA, and endotoxin and wherein at least one of thefirst and second pH is higher than the isoelectric point ofchondroitinase ABC.

Chondroitinase ABC is eluted in a linear gradient solution of pH 6-9NaCl to yield chondroitinase ABC at a purity of at least 98% and a yieldof at least 70% in a single pass of the heparin-immobilizedchromatography column.

In a further aspect, the washing liquid may be a pH 8-10 buffer.

In a further embodiment, endotoxin in the eluate of chondroitinase ABCmay be removed by filtration after elution, optionally to less than0.0002 EU/U.

In one aspect, the washing liquid may be a carbonate buffer, a phosphatebuffer, a borate buffer, a Tris-HCl buffer or a Tris acetate buffer.

In an embodiment, the pH of the matrix containing chondroitinase ABC ABCis adjusted to 7.0.

In one aspect, the conductivity of the matrix including chondroitinaseABC is adjusted to 1-5 mS/cm.

In a further aspect, the heparin is immobilized on a resin for proteinpurification having a hydroxyl group, an amino group and/or a carboxylgroup.

The resin may comprise one or more of agarose, cellulose, dextran,silica polyacrylamide or acrylic acid polymer.

In one aspect, the heparin is covalently bonded to the resin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a chromatogram of Example 2 of the present invention.

FIG. 2 shows a chromatogram of Example 3 of the present invention.

FIG. 3 shows an SDS-PAGE diagram of Example 3 of the present invention.

FIG. 4 shows a chromatogram of Example 4 of the present invention.

FIG. 5 shows an SDS-PAGE diagram of Example 4 of the present invention.

FIG. 6 shows a chromatogram of Example 5 of the present invention.

FIG. 7 shows an SDS-PAGE diagram of Example 5 of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel and simple method for thepurification of ChABC which can be applied to various ChABC-containingmatrices. As used herein, the term “matrix” refers to a mixture ofextracellular material or intracellular material (protein, DNA, lipid,etc.) from a host cell as a result of the host cell producing ChABC,along with the ChABC itself. The matrix may include bacteria, fungi,extracts containing surfactants, supernatants collected duringfermentation, and other materials from the production of ChABC from ahost material. The purification method is a chromatography method usingan affinity column capable of selectively capturing ChABC. The affinitycolumn is a column that binds to a specific ligand that specificallyinteracts with ChABC under chromatography conditions.

In an embodiment of the present invention, the ChABC, which isrepresented by the primary amino acid sequence of SEQ ID NO: 1, iseither obtained from Proteus vulgaris or from the cell of the hostmaterial including but not limited to Escherichia. coli, Bacillussubtillis, Proteus vulgaris, Pichia postoris, Saccharomyces cerevisiae,etc. which recombinantly expresses the protein. The ChABC expressed fromthe recombinant host cell without additional processing contains anadditional methionine (the amino acid sequence thereof is represented bySEQ ID NO: 2) at the N-terminal which is originated from the start codon“ATG”. The N-terminal methionine can be removed to generate the matureChABC with the amino acid sequence of SEQ ID NO: 1.

In the preparation of prokaryotic recombinant expression systems, thetarget gene for the expressed protein shall be cloned into an expressionvector or integrated into the host's chromosomal DNA. In order toexpress the gene, start codon shall be present in the 5′ end of thegene, which is translated into methionine at the N-terminal of theexpressed protein. In many cases, the mature protein expressed in theirnatural host are naturally processed by other enzymes, for example,signal peptidase, during the extracellular exportation. In other words,the N-terminal of many naturally processed proteins contain noN-terminal methionine. Without introducing proper processing machinery,the recombinant protein will contain an additional and unnaturalmethionine at the N-terminal.

In order to produce a native protein without an additional N-terminalmethionine, methionine aminopeptidase is usually cloned into theexpression host and co-expressed with the recombinant target protein.Methionine aminopeptidase can remove the N-terminal methionine, but thisenzyme has limited activity against the protein with charged or bulkyamino acid residue at the P1′ position. In addition, the activity ofmethionine aminopeptidase is dependent on the P2′ position of therecombinant protein. Another strategy of generating recombinant proteinwith native sequence without additional N-terminal methionine is byutilizing protease that can digest the protein specifically and have noor low selectivity on the P1′ position, i.e., without leaving a residueat the C-terminal side after the enzymatic digestion, after beingpurified from the host matrix. In this method, a DNA sequence encodingan affinity tag, e.g., maltose binding protein, His-tag, etc., are fusedto the 5′ end of the target gene. The protein expressed is thereforeunnaturally tag-fused. However, the tag can be specifically removed bycertain proteases, e.g., TEV protease, factor Xa, etc. Although this caneffectively generate recombinant protein without additional methionine,the enzymatic digestion is conducted in vitro after the proteinpurification, and an additional chromatographic step is required toremove the protease.

To solve this problem, in the present invention, an engineered TEVprotease that have a broad P1′ selectivity is cloned into the expressionhost and co-expressed with ChABC gene of which a TEV proteaserecognition site (amino acid sequence ENLYFQ) has been added between theinitial methionine and the native ChABC sequence.

For the recombinant bacterial expression system, the gene encoding theChABC protein (SEQ ID NO: 3) is ligated into an expression vectorcontaining promoter and Shine-Dalgarno (SD) sequence that can enablehigh transcription level in the host cell. Promoter of the vector usedin E. coli expression system can be T7, T5, Lac, Trp, araC or any hybridpromoter of them. Promoter of the vector used in B. subtilis can beSpac, SacB, Xyl, PBAD, Pgrac or any hybrid promoter of them. Apart fromtransforming the host bacteria with recombinant replicable expressionvector, chromosomal integration of ChABC gene into the host bacteria canbe another strategy for overexpression of ChABC. Some examples ofchromosomal integration vector include pSG1112 (Liu et al., 2004) forthe B. subtilis 1A304(Φ105MU331) and pMG1 (Gimpel et al., 2012) for B.subtilis MG1P.

The expression of ChABC from Proteus vulgaris can be induced byintroducing chondroitin sulfate, which is a known inducer for theproduction of the protein, into the culture medium through fed batchfermentation. In the recombinant expression system, the inducer isvector- and promoter-dependent, for example, IPTG for the lac promoter,arabinose for pBAD promoter. In the case of intracellularly expressedChABC, the bacteria can be harvested by centrifugation or cross-flowmicrofiltration, depending on the scale of the fermentation. Theextraction of ChABC from harvested bacteria can be either by mechanicaldisruption, for example, sonication, liquid homogenization, freeze-thaw,etc., or by chemical disruption, for example, introducing surfactant,lysozyme or any other lytic enzyme. ChABC in the extract or lysate ispurified by the affinity chromatography method of the present invention.

To determine a suitable affinity material the inventors selected a largenumber of different types of polysaccharide derivatives such asglycosaminoglycans or other polysaccharides such as alginates, fucoidan,polygalacturonic acid, pentosan polysulfates, cellulose phosphates, etc.for determination of suitability for use in affinity chromatography.Although immobilization of the selected polysaccharide on the resin isthe most direct way of testing the suitability of these ligands forChABC affinity chromatography, the immobilization, ligand density,spacer groups, etc. between the ligand and the resin surface cansignificantly affect the interaction between ChABC and the immobilizedpolysaccharides. In other words, the determination of the bindingbetween ChABC and polysaccharide in the solution phase is moreappropriate and accurate for revealing the intensity of the interaction.

There are several existing methods for quantitative study ofprotein-small molecule interactions such as differential scanningcalorimetry, quartz crystal microbalance, fluorescence anisotropy, andthe like. However, these methods require complex equipment, areexpensive and time consuming, requiring protein immobilization orprotein labeling. In order to save time and reduce costs,semi-quantitative assessment methods can be applied, such as enzymeinhibition assays. In the present invention, the relative bindingaffinity is evaluated by monitoring the inhibition of ChABC by theselected polysaccharide. The stronger the observed inhibitory effect,the higher the affinity between the ChABC and the polysaccharide. A moredetailed experimental setup is shown in Example 1. It was found thatheparin almost completely inhibited ChABC activity when an equal amountof substrate (chondroitin sulfate C) was administered by enzymeinhibition assay. Thus, heparin was identified as the most suitableligand among the selected screened polysaccharides for constructing theaffinity column.

In the present invention, heparin may be immobilized on different resinshaving a hydroxyl, amino, and carboxyl group for ligand immobilization.Heparin may be covalently bonded to the resin. For the resin, thepresent invention is not particularly limited as long as heparin can becovalently bonded to the resin. Selected resins include, but are notlimited to agarose, cellulose, dextran, silica polyacrylamide, oracrylic polymers. The heparin or resin may be activated by variousactivators including, but not limited to, cyanogen bromide, EDC/NHS(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/succinimide), sodiumperiodate, epichlorohydrin, and the like.

In the present invention, the affinity column to which heparin isimmobilized, particularly covalently bound to heparin, does not requirefurther coating. In particular, the column is not further coated withglycosaminoglycans which are degraded by ChABC. Optionally, furthercoating is feasible without the use of glycosaminoglycans.

Although heparin can bind and can significantly inhibit ChABC, theaffinity between immobilized heparin and ChABC in chromatography isunknown. To demonstrate that ChABC still has affinity for immobilizedheparin, the purified ChABC was loaded into a chromatography column ofheparin coupled via amide bonds to crosslinked agarose beads and washedwith the various buffers described in Example 2. In fact, Tris acetatebuffer (pH 9.2) that has a pH above the isoelectric point (pI=8.5) ofChABC was applied to the column. At the pI, ChABC becomes negative, andnegatively-charged ChABC and the negatively-charged heparin on the resinshould repel each other. If ChABC has only a non-specific chargeinteraction with heparin, it will elute from the column. However, fromthe experimental results, ChABC was not only successfully captured bythe resin, but was not eluted by a buffer having a pH above the pI. Inother words, ChABC has a specific interaction with heparin, rather thanpure nonspecific charge interactions.

In the present invention, the ChABC affinity chromatography column usingheparin has a significant advantage over the most commonly used cationexchange column for ChABC purification because of the significantlylower amount of impurities that are non-specifically bound to the resin.Thus, the affinity chromatography of the present invention can beapplied to various matrices with different impurity spectra including,but not limited to, Escherichia coli, Bacillus subtilis, Proteusvulgaris, Yeast (Pichia pastoris and/or Saccharomyces cerevisiae), toobtain highly purified ChABC. However, since heparin is a negativelycharged polysaccharide, a small amount of positively charged protein maystill be non-specifically captured by heparin immobilized resin.However, it was determined that these nonspecific binding proteins canbe removed by application of high pH buffers (pH 8-10). Selected pH 8-10buffers include carbonate buffer, phosphate buffer, borate buffer,Tris-HCl buffer or Tris acetate buffer, such as sodium carbonate buffer,sodium phosphate buffer, potassium carbonate buffer, potassium phosphatebuffer, sodium borate buffer, potassium borate buffer. Buffers in thispH range can significantly reduce nonspecific protein interactionsbecause most of the protein impurities will become negatively chargedand therefore do not interact with the heparin immobilized resin withcharge. The effect of ChABC is not affected by the high pH buffers.After removal of protein impurities, ChABC was eluted with a lineargradient of NaCl at pH 6-9. The eluate may contain ChABC (Examples 3-5)having a purity of up to 98-99% or more and having a low endotoxinlevel. The endotoxin can be further removed by passing the eluatethrough an endotoxin capture filter. In addition to low endotoxin andprotein impurities, another advantage of this process is simplicity andhigh recovery. Single-step chromatography significantly reduces theprocessing time, thus avoiding the loss of activity during processing.In fact, the final yield is in the range of 70-80% (Examples 3-5), whichis significantly higher than all existing methods. More importantly,affinity chromatography can be applied to different types of substratesproduced by different host cells.

Example 1

In the present invention, it is necessary to identify molecules thathave a similar structure to the substrate of ChABC but are inert toenzymatic degradation. In order to test polysaccharides that couldactually bind to the active site of ChABC, a relative enzyme inhibitionassay was conducted on the selected polysaccharide:

490 μL of the substrate solution was incubated at 37° C. for at least 15minutes containing 0.2% chondroitin sulfate C, 0.2% selectedpolysaccharide and 50 mM pH8 Tris HCl buffer. 10 μL of ChABC withactivity of about 100 U/L was incubated at 37° C. for at least 3minutes. After incubation, the two solutions were gently mixed. Themixture was then incubated at 37° C. for 20 minutes to perform enzymaticdegradation of chondroitin sulfate. After 20 minutes, the reaction wasterminated by inactivating ChABC by heating at 100° C. for 2 minutes. Inaddition to inactivating the mixture immediately without incubation for20 minutes, a blank control was performed in the same manner. Theabsorbance of the heat inactivated solution at 232 nm was then measuredand the apparent activity was calculated as shown in Equation 1. Theassay in the absence of selected polysaccharide was also conducted inthe same manner as a reference. The calculation of the percentage ofinhibition was showed in Equation 2:

$\begin{matrix}{{U\text{/}L} = {\frac{Abs}{E \times t} \times \frac{Vt}{Vs} \times 1000 \times {DF}}} & \left( {{Equation}\mspace{14mu} 1} \right)\end{matrix}$

where

-   -   Abs=Difference in absorbance        -   =OD232 nm in sample tubes−OD232 nm in blank tubes    -   E=Millimolar extinction coefficient of unsaturated disaccharides        from        -   Chondroitin Sulfate C=5.5    -   t=Reaction time=20 min    -   Vt=Total volume of the assay=0.5 mL    -   Vs=Volume of enzyme used=0.01 mL    -   DF=Dilution factor    -   1000=Convert U/mL to U/L

$\begin{matrix}{{\% \mspace{14mu} {in}} = {\frac{A({poly})}{A({ref})} \times 100\%}} & \left( {{Equation}\mspace{14mu} 2} \right)\end{matrix}$

where

-   -   % rA=percentage of residual activity    -   A(poly)=the activity presence of selected polysaccharide    -   A(ref)=the absorbance of the assay without the presence of        selected polysaccharide

Among all polysaccharides, alginate and polygalacturonic acid had nosignificant effect on enzyme activity, whereas heparin had the strongestinhibitory effect on ChABC. The results of the relative enzymeinhibition assays are shown in Table 1:

TABLE 1 Polygalacturonic Sample Reference Heparin acid Alginic acidActivity 121 5.1 117 119 (U/L) % rA NA 4.2 97 98

Furthermore, as incubation of ChABC alone with the heparin does not giveany change in absorbance, heparin is not subject to the enzymaticdegradation activity of ChABC. Therefore, immobilization of heparin as aligand for affinity purification of ChABC is the most suitable choiceamong the selected polysaccharide derivatives.

Example 2

To test whether ChABC has a specific interaction with immobilizedheparin, a column filled with heparin coupled via amide bonds tocrosslinked agarose beads was used. The column was pre-equilibrated with25 mM Tris-acetate (pH 7.3) before loading the sample onto the column.ChABC was dissolved in 25 mM Tris-acetate (pH 7.5) and loaded onto acolumn. (1) 25 mM Tris-acetate (pH 9.2), (2) 100 mM Tris-acetate (pH9.2), (3) 60 mM Tris-acetate (pH 8) and (4) 100 mM Tris-Acetate (pH7.5). None of these buffers elute ChABC from the column. And when the0-0.3M NaCl linear gradient is applied, ChABC can be eluted from thecolumn. The chromatogram is shown in FIG. 1.

Example 3

Gene encoding ChABC (SEQ ID NO: 1) from P. vulgaris was cloned byconventional PCR from the genomic DNA of P. vulgaris. This PCR productcontain the DNA sequence encoding ChABC (SEQ ID NO:1) was used as atemplate for the second round PCR during which it was decided to add theDNA sequence encoding MGSENLYFQ to the N-terminal of the ChABC, so thatthe DNA sequence encoding TEV protease-recognition-site contained ChABC(SEQ ID NO: 3) was generated. The PCR product was ligated via T4 DNAligase to linearized pET3a to form a recombinant plasmid with theencoding sequence of native ChABC. The recombinant plasmid was thenintroduced into E. coli strain BLR (DE3) by electroporation. Thetransformed E. coli was plated on LB agar plate containing 50 μg/mlampicillin to allow selection of colonies transformed with the plasmid.The selected single colony was inoculated into LB medium at 37° C. with50 μg/ml ampicillin for 18 hours as a pre-culture and this pre-culturewas transferred into another LB culture medium at 37° C. containing 50μg/ml ampicillin in the ratio 1:100. This other culture medium wasfurther incubated at 37° C. until the OD600 reached 0.8. IPTG at 1 mMwas added to the culture medium for inducing the production of ChABC toprogress further 4 hours. The cell mass was harvested by centrifugationand stored at −80° C.

10 g of recombinant E. coli cells were resuspended in 25 mM Tris HCl (pH7.0) and subjected to sonication for 5 minutes. Cell debris were removedfrom the suspension by centrifugation at 17000 g for 1 hour. Thesupernatant was collected and the pH was adjusted to pH 7.0 by theaddition of NaOH. The conductivity of the supernatant was adjusted to1.2 mS/cm by the addition of double deionized water.

The supernatant containing ChABC was subjected to 0.22 μm filtrationprior to loading onto a heparin immobilized column with pH 7.5 25 mMTris-acetate pre-equilibrated. The column was then washed with (1) 100mM Tris-acetate (pH 7.5), (2) 60 mM Tris-acetate (pH 8), (3) 100 mMTris-acetate (pH 9.2) Of ChABC was linearly eluted by 0-0.5 M NaCl. Thechromatogram and SDS-PAGE are shown in FIG. 2 and FIG. 3.

The degree of purification in the above method is shown in Table 2.

TABLE 2 Total activity (U) Recovery yield (%) Supernatant before 3034 —chromatography Flow through 5 — Wash 1 <1 — Wash 2 <1 — Wash 3 <1 —Eluate 2998 76

It can be seen that the method of this example is capable of obtainingChABC with a purity of >99%, a recovery rate of 76% and an endotoxinlevel below 0.0002 EU/U.

Example 4

Gene encoding ChABC (SEQ ID NO: 1) from P. vulgaris was cloned byconventional PCR from the genomic DNA of P. vulgaris. This PCR productcontain the DNA sequence encoding ChABC (SEQ ID NO:1) was used as atemplate for the second round PCR which was decided to add the DNAsequence encoding MGSENLYFQ to the N-terminal of the ChABC, so that theDNA sequence encoding TEV protease-recognition-site contained ChABC (SEQID NO: 3) was generated. The PCR product was ligated via T4 DNA ligaseto linearized pHT43 to form a recombinant plasmid with the encodingsequence of native ChABC. The recombinant plasmid was then introducedinto B. subtilis 168 by electroporation. The transformed B. subtilis wasplated on a 2×TY agar plate containing 5 μg/ml chloramphenicol to allowselection of colonies transformed with the plasmid. The selected singlecolony was inoculated into a sterile 2×TY medium at 37° C. with 5 μg/mlchloramphenicol for 18 hours as a pre-culture and this pre-culture wastransferred into another sterile 2×TY culture medium at 37° C.containing 5 μg/ml chloramphenicol in the ratio 1:100. This anotherculture medium was further incubated at 37° C. until the OD600 reached0.8. IPTG at 1 mM was added to the culture medium for inducing theproduction of ChABC to progress further 4 hours. The cell mass washarvested by centrifugation and stored at −80° C.

5 g of recombinant B. subtilis cells were resuspended in 25 mMTris-acetate (pH 7.0) incubated with 1 μg/ml lysozyme for 20 min at 30°C. The bacterial cells were further lysed by sonication for 5 minutes.Cell debris was removed from the suspension by centrifugation at 17000 gfor 1 hour. The supernatant was collected and the pH was adjusted to pH7.0 by introduction of NaOH. The conductivity of the supernatant wasadjusted to 1.6 mS/cm by the introduction of double deionized water.

The supernatant containing ChABC was subjected to 0.22 μm filtrationprior to loading onto a heparin immobilized column with pH 7.5 25 mMTris-acetate pre-equilibrated. The column was then washed with (1) pH7.5 25 mM Tris-acetate, (2) pH 8 60 mM Tris acetate, (3) pH 9 100 mMTris acetate. The ChABC on the column was eluted with a linear gradientof 0-0.5 M NaCl. The chromatogram is shown in FIG. 4 and FIG. 5. Thedegree of purification is shown in Table 3.

TABLE 3 Total activity (U) Recovery yield (%) Supernatant before 2610 —chromatography Flow through <1 — Wash 1 <1 — Wash 2 <1 — Wash 3 <1 —Eluate 1931 74

It can be seen that the method of this example is capable of obtainingChABC with a purity of >99%, a recovery rate of 74% and an endotoxinlevel below 0.0002 EU/U.

Example 5

The cell paste of 25 g of Proteus vulgaris was resuspended in 25 mMTris-acetate (pH 7.0) containing 2% Triton X-100 preheated at 37° C. for30 minutes. The suspension was stirred continuously at 750 rpm andincubated at 37° C. for 2 hours. After extraction, the suspension wasdiluted 2-fold with 4-6° C. high-purity water. The diluted suspensionwas then immediately clarified by high-speed centrifugation at 17700 gcentrifugation. The supernatant was further clarified by filtrationthrough a micropore with a pore size of 0.2 μm. The pH of the clarifiedsupernatant was adjusted to pH 7.0, the conductivity was adjusted to 4.8mS/cm, and then loaded onto the heparin immobilized column. The heparinimmobilized column was pre-equilibrated with pH 7.0 25 mM Tris-acetate.After the supernatant was completely loaded onto the column, the columnwas washed with (1) pH 7.0 25 mM Tris-acetate, (2) pH 8.0 60 mMTris-acetate and (3) pH 10 100 mM Tris-Acetate 9.2 to remove impuritiesthat include unwanted proteins, DNA, endotoxin. ChABC was eluted with alinear gradient of 0-0.5 M NaCl and tested for endotoxin levels below0.0002 EU/U. Purified chromatograms are shown in FIG. 6 and FIG. 7. Thedegree of purification is shown in Table 4.

TABLE 4 Total activity (U) Recovery yield (%) Supernatant before 2810 —chromatography Flow through <1 — Wash 1 <1 — Wash 2 <1 — Wash 3 <1 —Eluate 2248 81

It can be seen that the method of this example is capable of obtainingChABC with a purity of >99%, a recovery rate of 81% and endotoxin levelsbelow 0.0002 EU/U.

Those skilled in the art, with the guidance of the above teachings, maymake various modifications and variations with respect to the invention.It is therefore to be understood that the invention may be practicedotherwise than as specifically described herein without departing fromthe spirit of the invention as set forth in the claims.

1. A matrix of a material comprising one or more microorganisms toproduce chondroitinase ABC, the microorganisms selected fromgenetically-modified Escherichia coli, genetically-modified Bacillussubtilis, Proteus vulgaris, Pichia pastoris, and/or Saccharomycescerevisiae; and chondroitinase ABC produced by the microorganisms. 2.The matrix of a material of claim 1, wherein said matrix is a mixture ofextracellular or intracellular material from said one or moremicroorganisms.
 3. The matrix of a material of claim 1, furthercomprising extracts containing surfactants, supernatants collectedduring fermentation, and/or other materials from the production ofchondroitinase ABC by said microorganisms.
 4. The matrix of a materialof claim 1, wherein said chondroitinase ABC comprises a primary aminoacid sequence of SEQ ID NO:
 1. 5. The matrix of a material of claim 4,wherein said chondroitinase ABC further comprises a removable methionineat the N-terminal of the primary amino acid sequence, which isrepresented by SEQ ID NO: 2, such that no additional processing isrequired when said chondroitinase ABC is expressed in saidmicroorganisms.
 6. The matrix of a material of claim 5, wherein saidchondroitinase ABC further comprises a TEV protease recognition sitebetween the removal methionine and the primary amino acid sequence ofsaid chondroitinase ABC and co-expressed with said chondroitinase ABC insaid microorganisms.
 7. The matrix of a material of claim 1, whereinsaid microorganisms are transformed by an expression vector containingan encoding sequence of said chondroitinase ABC, a promoter, andShine-Dalgarno sequence that enables high transcription level in thetransformed microorganisms.
 8. The matrix of a material of claim 7,wherein said promoter comprises T7, T5, Lac, Trp, araC, Spac, SacB, Xyl,PBAD, Pgrac, or any combination thereof.
 9. The matrix of a material ofclaim 6, wherein said chondroitinase ABC is represented by SEQ ID NO: 3.10. The matrix of a material of claim 1, wherein said microorganisms areintegrated with the genes of said chondroitinase ABC into correspondingchromosome(s) of said microorganism for over-expression of saidchondroitinase ABC.
 11. The matrix of a material of claim 10, whereinsaid genes are integrated into the chromosome(s) of said microorganismvia a chromosomal integration vector comprising pSG1112 and pMG1. 12.The matrix of a material of claim 1, wherein said chondroitinase ABC isover-expressed by said microorganism in the presence of an inducer beingintroduced into a culture medium of said microorganisms through fedbatch fermentation.
 13. The matrix of a material of claim 12, whereinsaid inducer is chondroitin sulfate.